Free Radical Biology and Medicine 53 (2012) 1625–1641 Contents lists available at SciVerse ScienceDirect Free Radical Biology and Medicine journal homepage: www.elsevier.com/locate/freeradbiomed Review Article The mechanism of redox sensing in Mycobacterium tuberculosis Shabir Ahmad Bhat, Nisha Singh, Abhishek Trivedi, Pallavi Kansal, Pawan Gupta, Ashwani Kumar n Council of Scientific and Industrial Research, Institute of Microbial Technology, Chandigarh 160036, India a r t i c l e i n f o abstract Article history: Received 12 April 2012 Received in revised form 3 August 2012 Accepted 3 August 2012 Available online 11 August 2012 Tuberculosis epidemics have defied constraint despite the availability of effective treatment for the past half-century. Mycobacterium tuberculosis, the causative agent of TB, is continually exposed to a number of redox stressors during its pathogenic cycle. The mechanisms used by Mtb to sense redox stress and to maintain redox homeostasis are central to the success of Mtb as a pathogen. Careful analysis of the Mtb genome has revealed that Mtb lacks classical redox sensors such as FNR, FixL, and OxyR. Recent studies, however, have established that Mtb is equipped with various sophisticated redox sensors that can detect diverse types of redox stress, including hypoxia, nitric oxide, carbon monoxide, and the intracellular redox environment. Some of these sensors, such as heme-based DosS and DosT, are unique to mycobacteria, whereas others, such as the WhiB proteins and anti-s factor RsrA, are unique to actinobacteria. This article provides a comprehensive review of the literature on these redox-sensory modules in the context of TB pathogenesis. & 2012 Elsevier Inc. All rights reserved. Keywords: Redox homeostasis Redox sensing Virulence DosS DosT DosR WhiB3 Anti-s factors Serine–threonine kinases Metabolic flexibility Free radicals Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1626 The role of redox stress in tuberculosis pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1626 The role of ROS in tuberculosis pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1626 The role of hypoxia in latency and reactivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1626 The role of nitric oxide (NO) in tuberculosis pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1627 The role of acidic pH stress in tuberculosis pathogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1628 Mtb lacks classical redox sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1628 DosS and DosT: heme-based sensors of redox, hypoxia, NO, and CO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1629 The Dos regulon and the mycobacterial response to hypoxia, NO, and CO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1629 The molecular mechanism of redox and gas sensing by DosS and DosT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1631 WhiB proteins as iron–sulfur cluster-based sensors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1632 WhiBs as iron–sulfur cluster proteins with protein disulfide reductase activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1632 WhiBs as redox-responsive transcriptional factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1634 WhiB proteins as virulence and stress response factors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1634 Redox-regulated s factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1634 The role of serine–threonine kinases in redox homeostasis of mycobacterium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1636 Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1637 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1637 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1637 n Corresponding author. Fax: þ91 172 2690585. E-mail address: ashwanik@imtech.res.in (A. Kumar). 0891-5849/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.freeradbiomed.2012.08.008 1626 S.A. Bhat et al. / Free Radical Biology and Medicine 53 (2012) 1625–1641 Introduction Infectious diseases lead to the deaths of 15 million people annually and continue to be the primary cause of morbidity and mortality in underdeveloped and developing nations [1]. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), has latently infected one-third of the world’s population and remains the leading cause of death by a single infectious agent (1.7 million deaths annually). Of those infected with Mtb, only 5–10% develop active TB. The remaining individuals can control but not eliminate the infection. These infected individuals have a 10% lifetime risk of developing TB, but suppression of the immune system by aging, HIV infection, or immunosuppressive therapy may increase their risk to 10% annually. The genetic and mechanistic details of how Mtb switches from the persistent state, which can last for decades, to the replicative form are not well characterized and represent an important gap in our understanding of the biology of TB pathogenesis. Mtb faces a number of host-generated oxidoreductive stresses during its cycle of infection. These stresses are believed to be important for the transition between latency and reactivation. Mtb senses redox stress through diverse mechanisms and then exploits its metabolic flexibility to survive the stress and to initiate the genetic program for entering and establishing a dormant state. A better understanding of Mtb physiology, including the mechanistic details of its sensory pathways, is important for the development of better diagnostic tools, effective vaccines, and potent drugs. The role of redox homeostasis in TB pathogenesis has recently been reviewed elsewhere [2,3]; hence this review focuses on the redox-sensing mechanisms used by mycobacteria. The role of redox stress in TB pathogenesis During infection, mycobacteria are exposed to a number of redox stresses, such as reactive oxygen species (ROS), reactive nitrogen species (RNS), acidic pH, nutrient starvation, and hypoxia. Exposure of mycobacteria to redox stress triggers changes in metabolism and physiology that not only help it to survive inside the host, but also allow it to express virulence factors that ultimately lead to disease pathogenesis. The level of redox stress in the microenvironment surrounding Mtb plays an important role in determining whether the bacilli enter the nonreplicating persistent state or the replicative state. The role of ROS in TB pathogenesis The primary type of cells encountered by Mtb upon infection is the alveolar macrophage. Human alveolar macrophages employ a battery of enzymes to generate oxidative stress in an effort to eliminate the pathogen. Examples of these enzymes include NADPH oxidase (NOX), myeloperoxidase, catalase, and hydrolases. NOX is a multiprotein enzyme system with core components such as p40phox, p47phox, p67phox, p22phox, and gp91phox, as well as a number of regulatory components. NOX extracts electrons from NADPH and transfers them to oxygen to generate superoxide radicals using a low-potential b-type cytochrome [4]. Superoxide radicals leads to generation of other ROS such as H2O2, hypochlorite, and hydroxyl radicals. A number of mycobacterial species, including Mtb [5], are susceptible to the cidal and growth-inhibitory activities of ROS. ROS have the potential to damage a number of cellular components, including lipids, proteins, and DNA. The toxic effect of ROS is further compounded in Mtb by the absence of important DNA repair pathways such as mismatch repair [6,7]. Mtb is equipped, however, with a number of protective mechanisms and enzymes, including superoxide dismutase, catalase (KatG), alkyl hydroperoxidase (AhpC), and peroxiredoxins, to neutralize the redox stress generated by the macrophage cells [2]. In addition, the mycolic acid-rich cell wall of Mtb also protects against ROS. Mtb maintains its intracellular redox potential using mycothiol as an intracellular redox buffer [8]. Mycothiol is a conjugate of N-acetylcysteine with a pseudo-disaccharide of glucosamine and myoinositol [9]. Using these protective mechanisms in a regulated manner, Mtb can survive physiologically relevant levels of ROS [2]. The role of ROS in TB pathogenesis is highlighted by the observation that alveolar macrophages and blood monocytes obtained from active TB patients produce significantly reduced levels of ROS upon Mtb infection compared with cells from healthy individuals [10,11]. This decrease in ROS is related to the decreased activities of NADPH oxidase and the enzymes of the hexose monophosphate shunt [10]. Because only a small percentage of infected individuals develop active TB, it is possible that the extent of ROS production by alveolar macrophages upon Mtb infection could dictate the outcome of infection. Further evidence for a role of ROS in TB pathogenesis comes from the observation that NOX2 is essential for Toll-like receptor 2-dependent inflammatory responses and 1,25-dihydroxyvitamin D3-mediated antimicrobial activity against Mtb via cathelicidin expression [12]. These observations suggest that ROS not only have potential mycobactericidal activity but also are important for activation of the inflammatory/antimicrobial response of macrophages. Furthermore, ROS are important for modulation of apoptosis, which is emerging as a major pathway of Mtb clearance in vivo [13]. ROS induce apoptosis through activation of apoptosisregulating signal kinase 1. In line with this, the p47phox pathway of ROS generation is responsible for the induction of proinflammatory responses during TB [14,15]. Finally, the importance of ROS in human TB is indicated by the fact that patients with chronic granulomatous disease (with a genetic defect in ROS production) are susceptible to infection from various species of Mycobacterium [16–19]. These studies strongly suggest that levels of ROS in the host immune system play an important role in TB pathogenesis. The role of hypoxia in latency and reactivation TB primarily affects the lungs. The oxygen-rich environment of the lungs is important for the growth of bacteria and the establishment of productive infection. Oxygen is also required for luxuriant growth of Mtb in vitro. On the other hand, gradual depletion of oxygen helps Mtb transition into a nonreplicative, drug-unresponsive, persistent state, wherein the bacteria can survive for decades and could resume an active replicative state upon exposure to ambient levels of oxygen [20,21]. Exposure to hypoxia leads to a number of changes in the physiology of mycobacterial cells, including inhibition of DNA, RNA, and protein synthesis; variability in acid fastness; thickening of the cell wall; and unresponsiveness to anti-TB drugs such as isoniazid [21]. These changes are similar to changes in the metabolism of bacilli recovered from infected tissues [22,23] and suggest that hypoxia is a physiologically relevant stress that modulates the outcome of the infection. Interestingly, during the preantibiotics era, treatment of TB included patient care at sanatoriums, therapeutic pneumothorax, thoracoplasty, and surgical removal of infected tissue. Many of these methods involved reduction of oxygen tension in the lung, thereby potentially initiating dormancy in Mtb and relieving the symptoms of TB. As an example, treatment of TB was primarily done at the sanatoriums. Most of the sanatoriums were constructed at high altitudes, where oxygen tension is low [24]. S.A. Bhat et al. / Free Radical Biology and Medicine 53 (2012) 1625–1641 The low oxygen tension at high altitude could be one of the underlying causes of relatively infrequent occurrence of active TB among people living at high altitudes [25–27]. In the preantibiotics era, pneumothorax was used as one of the ways to cure TB. In therapeutic pneumothorax, air is artificially introduced into the pleural cavity, causing partial collapse of a lung and leading to reduced oxygen tension in the surrounding region [28]. Moreover, therapeutic pneumothorax often leads to fibrosis around the diseased tissue and thus containment of the infection, as seen with closed tubercular cavities potentially harboring dormant Mtb. Similar to therapeutic pneumothorax, thoracoplasty also involves surgery of the thorax for TB treatment [29]. In thoracoplasty, ribs from one side of the thorax are bent or removed to collapse the infected portion of lung permanently. Therapeutic pneumothorax and thoracoplasty were recently used successfully for treatment of multidrug-resistant and extensively drugresistant cases of TB, further emphasizing the role of oxygen tension in mycobacterial growth and latency [30–32]. More evidence that oxygen tension is involved in latency comes from the observations that postprimary TB is mainly associated with the upper lobe of the lung (the most oxygen-rich tissue of the body) [33,34] and that actively replicating bacteria are recovered from the lesions exposed to air, whereas less bacterial load is observed in lesions lacking direct contact with oxygen [35]. In addition, human granulomas are often avascular, indicating that Mtb faces hypoxia in humans [36]. Consistent with this hypothesis, granulomas of nonprimates, rabbits, and guinea pigs are also hypoxic [37]. These observations strongly suggest that the levels of oxygen in the granuloma dictate whether the bacteria will actively replicate to cause active TB disease or enter a state of nonreplicative persistence (Fig. 1). Because the transition from replicative state into nonreplicative state requires synchronized regulation of Mtb metabolism, Mtb must employ a battery of versatile sensors to continuously monitor the levels of oxygen in its microenvironment. The role of nitric oxide (NO) in TB pathogenesis Similar to hypoxia, exposure to NO is believed to be a physiologically relevant signal for initiating mycobacterial persistence. Low concentrations of NO inhibit Mtb respiration [38], leading to inhibition of anabolic processes such as DNA, RNA, and protein synthesis and thus inhibiting Mtb growth and promoting the drug-unresponsive persistent state [38]. Moderate and high concentrations of NO have bacteriostatic and bactericidal effects on Mtb, respectively [39,40]. Furthermore, exposure of Mtb to NO induces a genetic response similar to the one initiated by hypoxia [38,41,42]. A significant body of evidence demonstrating the importance of NO in TB pathogenesis has emerged from the murine model of TB. Murine macrophages produce bactericidal levels of NO upon activation with appropriate stimuli and are important for the control of TB [43]. The inhibition of exponential replication of mycobacteria in the murine model depends on the presence of inducible NO synthase (iNOS) [44] and inhibition of iNOS during the chronic phase of infection leads to exacerbation of the infection and death of the infected animal. In addition, iNOSdeficient mice are more susceptible to intravenous [44] and aerogenic infection [45]. Intriguingly, the presence of NO is essential for maintaining a latent TB infection in the murine model [46], suggesting a critical role for NO in TB dormancy. Infection Active Tuberculosis Latent Tuberculosis Hy pox i a/ E fficie nt imm un 1627 S e response/ ROS/ RN / An s rug Bd ti-T B-cells T-cells Extracellular mycobacteria Foamy macrophages Infected macrophages Ruptured macrophages Intracellular mycobacteria Fibroblast Collagen fibres Fig. 1. TB pathogenesis. Upon infection with bacilli residing in aerosols,  5–10% of individuals develop active tuberculosis disease, whereas 90–95% remain latently infected. The hallmark of tuberculosis disease is granuloma formation. The outcome of the Mtb infection is guided by the prevailing redox state of the environment in the granuloma. The structure of granulomas in active TB patients and in latently infected individuals is depicted. The latent infection is favored by hypoxia, efficient immune response, anti-TB drugs, ROS/RNS, and also by surgical interventions such as therapeutic pneumothorax or thoracoplasty. The reactivation of latent infection is assisted by immunosuppression through HIV infection or administration of immunosuppressive drugs, nutrient starvation, and hyperoxia. 1628 S.A. Bhat et al. / Free Radical Biology and Medicine 53 (2012) 1625–1641 The precise role of NO in human pulmonary TB remains elusive, because of a lack of vital resources and reagents such as a human alveolar macrophage cell line. However, significant evidence points to the importance of NO in human TB. Mtb infection of peripheral blood-derived monocytes or human monocyte cell lines leads to induction of iNOS and thus increased NO; this increase in NO is important for inhibition of Mtb growth [47]. More significantly, pulmonary macrophages from healthy individuals produce inhibitory concentrations of NO upon mycobacterial infection [48–50]. iNOS, endothelial NOS, and nitrotyrosine are detected in surgically resected lung tissue from patients with active pulmonary TB, suggesting a role for iNOS in human pulmonary TB [51]. Furthermore, TB patients exhale higher levels of NO than healthy control subjects do and their alveolar macrophages exhibit higher levels of iNOS activity [52]. Exhaled NO has recently been proposed as a potential diagnostic marker of TB [53]. In mice, iNOS is induced by interferon-g (IFN-g). In humans, mutations in the IFN-g receptor [54] and polymorphisms of the IFN-g promoter [55] are manifested as susceptibility to mycobacterial infection, further emphasizing the role of NO in TB pathogenesis. Arginine is administered as a dietary supplement that promotes increased NO and thus has the potential to improve the clinical outcome of active TB [56]. Furthermore, 1,25-dihydroxyvitamin D3 in the presence of IFN-g leads to induction of iNOS and reduces the growth of mycobacteria in human cells [57,58]. In addition to NO being mycobactericidal, it is also a secondary messenger that can modulate the adaptive immune system and function of phagocytic cells to affect the outcome of infection. This aspect of NO’s role in TB pathogenesis, however, has not been well studied. It was shown recently that NO induces apoptosis of macrophage cells to restrict the growth of Mtb [59]. Furthermore, NO produced by mesenchymal stem cells recruited at the site of infection can suppress the T cell response [60]. Together, these results suggest that iNOS-generated NO at higher concentrations could kill mycobacterial cells, whereas at the lower concentration it promotes the nonreplicative persistent state of Mtb by induction of the Dos regulon. Thus sensing, adaptation, and escape from the bactericidal activity of NO play a crucial role in TB pathogenesis. However, the molecular mechanisms of NO sensing and adaptation remain poorly understood. The role of acidic pH stress in tuberculosis pathogenesis Mtb is an intracellular pathogen that resides primarily in the phagosomes of alveolar macrophages. The pH of phagosomes in naı̈ve macrophages ranges from 6.3 to 6.5 [61], whereas the pH of activated macrophages ranges from 4.5 to 4.8 [62]. To establish a productive or latent infection, Mtb must survive the acid stress manifested by macrophages. To survive inside phagosomes and phagolysosomes, Mtb inhibits the acidification of phagosomes by producing and secreting ammonia [63] or by inhibiting vacuolar ATPase through secretory factors [64]. Mtb must also adjust its metabolism to maintain its internal pH despite fluctuations of the pH in its microenvironment [65]. A coordinated transcriptional response is observed when Mtb is exposed to pH values encountered inside naı̈ve or activated macrophages [66]. A number of in vitro and in vivo studies indicate that Mtb can resist significant levels of acidic pH stress and that its pH response is not similar to the classical acid tolerance phenotype of enteric bacteria [67]. In the classical acid tolerance phenotype, bacteria can resist exposure to highly acidic conditions after being primed with mildly acidic conditions [67]. The hypothesis that Mtb is exposed to acidic pH in human pulmonary TB is also supported by the fact that the frontline anti-TB drug pyrazinamide is activated only under acidic conditions [68]. These observations suggest that acidic pH plays an important role in the TB pathogenesis; however, the genetic mechanisms of pH sensing and tolerance are not known. Mtb lacks classical redox sensors The above-cited literature suggests that Mtb is exposed to a number of redox stresses such as ROS, hypoxia, NO, and acidic pH during its cycle of infection. The success of Mtb as a human pathogen indicates that it can perceive these stresses in the surrounding environment and modulate its metabolism to maintain its intracellular redox state. A number of classical sensors are employed by various species of bacteria to sense redox stress and hypoxia. These sensors include OxyR of Salmonella; FixL of Rhizobium; SoxR, fumarate/nitrate reduction regulator (FNR), and ArcB of Escherichia coli; and RexA of Streptomyces. OxyR regulates the transcriptional response of bacteria to peroxide stress. The OxyR regulon includes several of the oxidative stress enzymes such as KatG, alkyl hydroperoxide reductase, thioredoxins, glutaredoxins, and the enzymes of the iron–sulfur cluster biosynthetic pathway [69,70]. OxyR harbors six Cys residues, of which Cys199 and Cys208 are redox reactive [71]. Under nonoxidizing conditions, Cys199 remains buried in a hydrophobic cavity and interacts with neighboring Arg266. This interaction renders Cys199 oxidation sensitive. In the presence of oxidative stress, Cys199 is oxidized to sulfenic acid and comes close to Cys208, thus facilitating disulfide bond formation [72]. The tetrameric form of oxidized OxyR can activate the transcription of the OxyR regulon. Upon establishment of a reducing intracellular environment, the oxidized OxyR can be reduced back to the transcriptionally inactive form by glutaredoxins [71]. The other classical sensors include the heme-based oxygen sensor FixL, iron–sulfur cluster-based sensors SoxR and FNR, and the NADH/NAD þ sensor RexA. FixL is a heme-based sensor of Rhizobium meliloti that regulates the expression of genes involved in nitrogen fixation in response to the presence of oxygen [73]. The binding of O2 to the heme of FixL inhibits the kinase activity of FixL, whereas in the absence of O2, the heme remains in the deoxy form, which possesses increased kinase activity [74]. SoxR is an iron–sulfur cluster-based sensor that regulates the transcriptional response of E. coli to the superoxide radical. SoxR contains a [2Fe–2S] cluster [75]. Under normoxic conditions, the SoxR iron–sulfur cluster is maintained in the [2Fe–2S]1 þ state; upon exposure to superoxide radicals, it is oxidized to [2Fe–2S]2 þ [76]. This transition initiates transcription by SoxR [77]. FNR is also an iron–sulfur cluster-based redox sensor. However, it regulates components of an alternative electron transport chain through a different mechanism. Under anaerobic conditions, the FNR iron–sulfur cluster is cubic, [4Fe–4S]2 þ , and upon exposure to oxygen, it is converted into the planar [2Fe–2S]1 þ [78]. This switch of oxidation states is associated with conversion of transcriptionally active, dimeric FNR into inactive, monomeric FNR [79]. RexA, in Streptomyces coelicolor, is another unique sensor of intracellular redox status. RexA harbors a Rossmann fold capable of binding pyridine nucleotides at the C terminus, whereas the N terminus acts as a DNA-binding domain. Both NAD þ and NADH can bind to the C-terminal sensory fold of Rex, but only NADH binding decreases the affinity of RexA for the promoters of the cydABCD and hemACD operons and leads to reduced expression [80]. The decreased flow of electrons in the electron transport chain (ETC) caused by the unavailability of oxygen during hypoxia leads to accumulation of NADH. On the other hand, ArcB monitors the redox state of the cell by sensing the levels of reduced ubiquinones in the ETC [81]. Under hypoxic conditions, inhibition of respiration leads to accumulation of the reduced form of S.A. Bhat et al. / Free Radical Biology and Medicine 53 (2012) 1625–1641 ubiquinones. Accumulated ubiquinol initiates the formation of interchain disulfide bridges in the ArcB dimer. The disulfide bond formation leads to inhibition of the kinase activity of ArcB [82]. Bacteria are persistently exposed to organic hydroperoxides (OHPs) resulting from oxidation of unsaturated fatty acids by molecular oxygen or ROS. Bacteria employ a family of proteins known as organic hydroperoxide resistance protein (Ohr) to detoxify the OHPs [88]. The expression of Ohr is under the control of an organic hydroperoxide sensor, OhrR (organic peroxide resistance regulator) [89]. OhrR acts as a repressor of ohr. OhrR harbors a redox-active conserved cysteine that reacts with the OHP to generate a sulfenic acid intermediate. This sulfenic acid derivative of OhrR could react with the low-molecular-weight thiol leading to formation of a mixed-disulfide species of protein that is incapable of binding to DNA. This mixed-disulfide species could be reconverted to the thiol form upon reduction through a thiol–disulfide exchange reaction. This reduction leads to activation of DNA binding activity of OhrR [90–92]. A homolog of Ohr has been identified and characterized in Mycobacterium smegmatis (MSMEG_0447) by exploiting the transposon mutagenesis of a strain lacking mycothiol (MSH) [93]. This strain overproduced Ohr to compensate for the lack of MSH [93]. However, a homolog of ohr could not be detected in pathogenic mycobacteria [93]. The absence of Ohr in the Mtb genome could be one of the underlying reasons for its increased sensitivity toward the loss of MSH and organic hydroperoxides compared to M. smegmatis [93]. Although Mtb lacks the ohr gene, it possesses another protein (Rv2923c) of the OsmC protein subfamily previously known for the response to osmotic stress. This protein has a higher degree of similarity with Ohr proteins. Rv2923c and its homolog in M. smegmatis (MSMEG_2421) have been shown to possess organic hydroperoxide reductase activity. Unlike Ohr proteins, OsmC proteins can reduce hydrogen peroxide as well [94]. Identifying a regulator of osmC could lead to the identification of a peroxide sensor in mycobacteria. MgrA is another sensor protein belonging to the MarR family of proteins possessing a helix–turn–helix (HTH) domain and which bind to DNA. This regulator protein regulates autolysis, virulence genes, and efflux pumps and is also involved in biofilm regulation [95–97]. Similar to OhrR, MgrA harbors a redox-active cysteine that regulates its DNA-binding activity [98,99]. This conserved cysteine could be oxidized by various species of ROS including hydroperoxide and organic peroxide. This oxidation leads to conformational changes in the structure of MgrA, decreasing its affinity for DNA [98,99]. Interestingly, PknB of Mtb has significant similarity to MgrA, but its function as a redox sensor has not been analyzed. In E. coli and Salmonella, pretreatment with mild oxidative stress provides protection from stringent oxidative stress. This protective response comes from the presence of redox sensors such as OxyR. A similar protective response is detected only in the saprophytic species of mycobacteria, such as M. smegmatis, and is absent from the pathogenic, slow-growing species of mycobacteria, including Mtb [83], suggesting that Mtb lacks the classical sensors of oxidative stress. A more detailed analysis suggests that the genetic locus coding for divergent expression of OxyR and AhpC is conserved in all species of mycobacteria, but the OxyR of pathogenic, slow-growing mycobacterial species is inactivated by multiple mutations [84]. Sequencing of the Mtb genome revealed a putative cyclic AMP receptor protein (Crp)/FNR homolog encoded by open reading frame Rv3676. This protein shares 32% sequence identity with E. coli Crp and is devoid of cationic metal ion [85]. It is a transcription regulator that is regulated by cellular cAMP levels [86]. It plays an important role in bacterial survival in the mouse TB model and regulates the expression of resuscitation-promoting factor [87]. Despite the homology to FNR, Mtb lacks a true [Fe–S] cluster-based FNR. 1629 Using the published genome and proteome sequences of Mtb, other homologs of classical redox sensors could not be detected. Recently published work by Voskuil et al. suggests that exposure to different levels of oxidative stress mediated by different oxidizing agents such as H2O2 and NO results in different transcriptional responses [100]. A number of uncharacterized putative redox regulators were also induced as a part of the transcriptional response to oxidative stress. However, the redoxsensing/regulating mechanism for these and many other uncharacterized sensors/regulators in the biology of Mycobacterium remains to be analyzed. The protective response conferred by Mtb despite the absence of classical redox sensors suggests that Mtb could utilize novel sensors to perceive the surrounding microenvironment. The discovery of mycobacterial redox sensors and their characterization was begun only recently and is believed to be an area of scientific interest in which coming years will see the characterization of more redox sensors and their importance in TB biology. A number of recently published elegant studies have established an important role for some of the recently characterized redox regulators in the virulence and pathogenesis of tuberculosis. A recently published review has described the role of redox signaling in human pathogens [99]; hence a detailed description of the current understanding of mycobacterial redox sensors is presented in the following sections. DosS and DosT: heme-based sensors of redox, hypoxia, NO, and CO Although Mtb lacks classical redox sensors such as FixL, OxyR, and FNR, it possesses novel heme-based sensors in the form of DosS and DosT. DosS and DosT, along with DosR, constitute the DosRST two-component system, wherein DosS and DosT act as sensor histidine kinases that sense the presence or absence of ligands (that may also act as an oxidant) and relay it to the response regulator DosR (Fig. 2). The Dos regulon is believed to play an important role in the transition of Mtb from the actively replicating state to the nonreplicating, persistent state. The following sections describe the molecular mechanism of redox and gas sensing by DosS and DosT and the role of the Dos regulon in Mtb physiology. The Dos regulon and the mycobacterial response to hypoxia, NO, and CO The potential role of hypoxia in human TB and Mtb latency has prompted researchers to invest significant effort toward understanding the effect of hypoxia on the physiology of Mtb. Mtb is aerobic and sudden depletion of oxygen is lethal [101], but gradual depletion of oxygen initiates a phenotypic switch in Mtb that enables it to survive for decades [20,21]. Upon exposure to hypoxia in vitro, Mtb becomes elongated, its cell wall becomes thick, the acid-fast character of Mtb is lost, and it becomes unresponsive to anti-TB drugs [21,102]. These phenotypic characteristics are very similar to the distinctive attributes of latent Mtb, suggesting that hypoxia plays an important role in Mtb latency. In general, hypoxia reduces the flow of electrons in the ETC because of the unavailability of oxygen as a terminal electron acceptor. This decreased flow of electrons leads to an accumulation of NADH, reduced ubiquinones, and reduced cytochromes in the bacteria. Survival of bacteria during hypoxia depends on induced expression of an alternative cytochrome oxidase (cytochrome BD oxidase) with higher affinity for oxygen, changeover from proton-pumping NADH dehydrogenase I to non-protonpumping NADH dehydrogenase II, and a number of other important 1630 S.A. Bhat et al. / Free Radical Biology and Medicine 53 (2012) 1625–1641 Fig. 2. Sensing mechanism of DosS/DosT. (A) The role of the Dos regulon. DosR regulates  50 genes that are collectively known as the Dos regulon. The Dos regulon is activated under hypoxic and reductive conditions and in the presence of CO and NO. The Dos regulon includes genes encoding the nitrate–nitrite antiporter (NarK2) and nitrate reductase (NarX). These proteins facilitate the survival of Mtb in hypoxia and acidic pH and its in vivo persistence. DosR also regulates expression of the fumarate reductases (frdABCD), ferridoxin (fdxA), and formate-hydrogen lyase systems that act as alternate electron transfer systems in the absence of oxygen. Furthermore DosR regulates other physiological responses such as DNA biosynthesis, triacylglycerol (TAG) biosynthesis, and growth of Mtb by regulating the expression of ribonucleoside diphosphate reductase (nrdZ), triacylglycerol synthase (tgs1), and Rv2633, respectively. (B) Mechanism of redox/gas sensing by DosS. The kinase activity of DosS is regulated by the ligation/redox state of the heme iron. Under hypoxic conditions the heme iron of DosS is in the deoxy ferrous (Fe2 þ ) form and the DosS protein is kinetically active. Binding of NO or CO to heme iron does not inhibit the kinase activity of DosS. However, under normoxic conditions the DosS heme iron either binds oxygen or becomes oxidized. This binding of oxygen or oxidation of heme iron leads to inhibition of kinase activity. The redox sensing model proposes another level of regulation by unknown factors that could reduce the Fe3 þ heme iron into the Fe2 þ form. (C) Mechanism of gas sensing by DosT. Unlike the redox sensing mechanism of DosS, DosT heme iron binds oxygen in a concentration-dependent manner. This binding of oxygen leads to inhibition of the kinase activity of DosT. However, similar to DosS, the binding of NO and CO could lock DosT in the active conformation. S.A. Bhat et al. / Free Radical Biology and Medicine 53 (2012) 1625–1641 metabolic changes. These alterations help the bacteria to minimize the reductive stress generated from the accumulation of reduced carriers of electrons. Bacteria have evolved sensors such as ArcB of E. coli to sense the depletion of oxidized ubiquinones and RexA of S. coelicolor to sense NADH/NAD þ . To understand Mtb’s response to hypoxia, Sherman et al. exposed Mtb cells to defined hypoxia and analyzed the transcriptome using microarray technology [103]. This study demonstrated that, similar to E. coli and other bacteria, Mtb responds to hypoxia by inhibiting overall biosynthesis of RNA and protein. Approximately 50 genes were upregulated in response to hypoxia (Fig. 2A). This transcriptional response was termed the Dos (dormancy) regulon [104]. The Dos regulon contains a paired two-component system called DosRS, which, via phosphorylation and activation of DosR by DosS, regulates the DosR regulon [42,103]. DosT (Rv2027c), an orphan histidine kinase and a homolog of DosS, can also specifically phosphorylate DosR [105,106]. Unlike DosS, which is under the control of DosR, DosT is constitutively expressed and is not regulated by DosR. As with exposure to hypoxia, exposure of Mtb to NO inhibits the respiration of Mtb, restricts its growth, and specifically induces the Dos regulon [38]. Furthermore, we and others [107,108] have demonstrated that exposure to CO also induces the Dos regulon. Interestingly, Mtb infection of naı̈ve macrophages induces the expression of heme oxygenase 1 (HO-1), independent of IFN-g or iNOS [107]. HO-1 catalyzes the oxidative degradation of heme and leads to formation of biliverdin, molecular iron, and CO. Using bone marrow-derived macrophages from HO-1 knockout mice, we [107] and others [108] have demonstrated that physiological levels of CO lead to the induction of the Dos regulon in naı̈ve macrophages. Under hypoxic or anoxic conditions, bacteria must use alternate electron acceptors such as nitrate or fumarate for continued production of ATP through the ETC. For example, E. coli reduces nitrate to nitrite through the nitrate reductase complex using electron flux from the ETC. In E. coli, this system requires a nitrate–nitrite antiporter that is involved in the uptake of nitrate and the export of nitrite that results from nitrate respiration [109]. The level of nitrate reductase complex is not transcriptionally regulated; instead, the expression of the transporter is regulated and is induced upon hypoxia [110]. Similar to E. coli, Mtb can use alternate electron acceptors in the absence of oxygen. A number of enzyme systems required for continuing the ETC in the absence of oxygen are under the control of the Dos regulon. These systems include nitrate reductase, fumarate reductase, formate-hydrogen lyase, and ferridoxins. DosR regulates the expression of the nitrate–nitrite antiporter narK2 along with the fused nitrate reductase narX. This system is important for the survival of Mtb in hypoxia [111] and acidic pH [112] and for in vivo persistence [113]. These observations suggest that DosRregulated nitrate respiration is used by Mtb for survival in vivo and could be important for transition into latency. The other preferred alternate terminal electron acceptor is fumarate. Fumarate can be reduced to succinate by fumarate reductase using the flux of electrons from the etc. Indeed, increased levels of succinate are secreted by Mtb exposed to hypoxia [114]. The expression of fumarate reductase (frdABCD) is controlled by DosR. Furthermore, DosR regulates the expression of fdxA, an alternate electron transfer protein that may play an important role in electron transfer reactions during adaptation to hypoxia. Interestingly, DosR also regulates the expression of enzymes encoding the formate-hydrogen lyase (FLH) system. The FLH system converts formate into H2 and CO2 using two enzymes: formate dehydrogenase-H and hydrogenase-3 [115]. This system functions in the absence of oxygen and other alternate terminal electron acceptors. 1631 In addition to regulating alternate electron acceptors, DosR also regulates a number of important pathways that help Mtb adapt during stress conditions. In a series of elegant experiments, Leistikow et al. demonstrated that DosR is essential for maintaining energy levels and redox balance during the downshift to the nonreplicating state upon anaerobiosis [116]. Additionally, the Dos regulon is critical for resumption of growth upon the return of aerobic conditions. DosR governs DNA biosynthesis, triacylglycerol biosynthesis, and general stress response upon exposure to hypoxia and NO [116]. It regulates the expression of ribonucleosidediphosphate reductase (nrdZ), which catalyzes the biosynthesis of deoxyribonucleotides from the corresponding ribonucleotides using the reductive energy provided by thioredoxins. DosR also regulates tgs1, which encodes triacylglycerol synthase 1 (Tgs1), one of the most active of 10 triacylglycerol synthases encoded by the Mtb genome. It was recently demonstrated that triacylglycerol biosynthesis plays an important role in reducing the growth rate of Mtb by redirecting cellular carbon fluxes away from the tricarboxylic acid cycle [117]. tgs1 mutants cannot arrest their growth, they consume oxygen faster than wild-type Mtb, and they show inefficient prolonged survival in the absence of oxygen. DosR regulates approximately eight universal stress proteins, including Rv2623. Rv2623 is believed to regulate TB latency; an Rv2623-deficient mutant fails to restrict growth in response to the adaptive immune response and thus cannot establish chronic infection in guinea pig and mouse models of TB [118]. Because of unrestricted growth, Rv2623-deficient Mtb strains exhibit a hypervirulent phenotype. These observations suggest that DosR plays an important role in Mtb’s adaptation to hypoxia. The molecular mechanism of redox and gas sensing by DosS and DosT DosS and DosT are sensor histidine kinases that relay signals to the response regulator DosR. They are equipped with a sensory domain and an activator/transmitter domain. The sensory domains contain two tandem GAF domains, so-named because of their characteristic presence in sensor proteins, namely, cGMPspecific phosphodiesterases, adenylyl cyclases, and FhlA. The transmitter domain of both sensors has a histidine kinase domain and an ATPase domain. Sardiwal et al. have demonstrated that the first GAF domain of DosS (GAF-A) covalently binds heme at His149 [119]; these proteins are the first examples of GAF-based proteins capable of binding heme. The unique coordination/redox chemistry of this heme allows DosS and DosT to modulate their kinase activity in response to variable levels of O2, NO, and CO. In 2007, the biochemical properties of DosS and DosT were independently characterized by us and others [120–122]. These studies showed that DosS and DosT bind heme type B. When oxygen binds to the heme of the DosS and DosT sensory domains, a signal is relayed to the activator/transmitter domain that results in the inhibition of the kinase activity of DosS and DosT (Fig. 2B and C). NO and CO binding do not inhibit the kinase activity; in contrast, these studies suggest that NO and CO can form complexes with the heme that could potentially lock DosS and DosT in their active states and lead to activation of the Dos regulon even in the presence of O2. A distal tyrosine (Tyr171) and the second GAF domain have been implicated in ligand discrimination [123,124]. Despite remarkable similarities between DosS and DosT, there are also a number of critical biochemical differences. For example, DosS and DosT have different binding affinities for gaseous ligands [122]. Furthermore, DosT is extremely resistant to oxidation [121,122], whereas DosS either is readily oxidized by O2 [121] or forms an unstable oxy complex [120,122,123]. The rate of DosS oxidation and whether oxygen makes a stable complex with the ferrous form of heme iron remain controversial (Fig. 2B). Our group has used absorbance spectroscopy and electron paramagnetic 1632 S.A. Bhat et al. / Free Radical Biology and Medicine 53 (2012) 1625–1641 resonance (EPR) spectroscopy to demonstrate that in the absence of O2, the heme iron of DosS exists in the deoxy ferrous form (Fe2 þ ). Upon exposure to O2, the heme iron is rapidly oxidized to the ‘‘met’’ (Fe3 þ ) form [121]. We have also shown that the met form of the protein is kinetically inactive. In addition, oxidizing agents other than O2, such as ferricyanide, can convert the active ferrous heme of DosS into the inactive met form [121]. Thus, DosS can be classified as a redox sensor. A different interpretation of kinase activity of DosS comes from Sausa et al. [122], Yukl et al. [123], and Ioanaviciu et al. [120]. These groups performed elegant experiments using absorbance spectroscopy and resonance Raman spectroscopy to demonstrate that DosS can form a relatively stable oxy form (Fe2 þ –O2). Moreover, they also demonstrated that the oxy form of DosS has NO dioxygenase activity similar to NO-detoxifying bacterial flavohemoglobins, further emphasizing the existence of a stable oxy–heme complex of DosS [125]. These results have led them to conclude that DosS is an oxygen sensor rather than a redox sensor. Our model of DosS as a redox sensor is supported by Cho et al. [126], who solved the crystal structures of oxidized, reduced, and reduced-then-air-oxidized complexes of the DosS GAF-A domain. They demonstrated that the met form of the GAF-A domain harbors a hexacoordinated heme ligated to His149 at the proximal end and a water molecule at the distal end. The reduced protein has a pentacoordinated heme that forms the met complex upon exposure to oxygen. Furthermore, this met heme in the GAF-A domain can be reduced by FADH, suggesting the possibility that an FADH-binding protein may control the rate of DosS reduction. Cho et al. further observed that heme inside the GAF-A domain has a restricted environment that does not permit a stable oxy– heme complex. Instead, a prevalent network of hydrogen bonds involving multiple water molecules and the side chains of several amino acids facilitates transfer of electrons from oxygen to heme [126]. These findings were further extended by a comparison of the crystal structures of the DosS and DosT GAF-A domains [127]. Whereas DosT has a wide-open channel that facilitates formation of the oxy–heme complex, the DosS GAF-A channel is narrow and closed by the carboxylate group of Glu87, which obstructs the access of O2 to the heme iron. These observations were further supported by the experiments in which replacement of Glu87 in DosS with Ala or Gly resulted in a GAF-A domain structure that allowed stable oxy–heme complex formation. In contrast, a conversion of glycine to glutamate at position 87 in DosT GAF-A rendered DosT oxidation sensitive [127]. The redox sensor model of DosS was further supported by elegant in vivo studies performed by Honaker et al. [128]. This group showed that a reduced electron transport system in the presence of ambient oxygen leads to upregulation of the Dos regulon through DosS; these results confirm that DosS is a redox sensor. They also observed that DosT does not respond to the reduced electron transport system; these results further confirmed that DosT is an oxygen sensor. The ETC was more reduced during hypoxic conditions; they reasoned that the reduced ETC is responsible for hypoxic induction of the Dos regulon in the DosT mutant. To further investigate, they inhibited oxidoreductases or the synthesis of menaquinone and observed that in both cases, the extent of Dos regulon induction was diminished [128]. The above discussion details the controversy surrounding the identity of DosS as a redox sensor or as an oxygen sensor. The underlying causes of these divergent results are unclear and indicate the difficulties associated with working on these oxygen-sensitive proteins. Despite controversy over the mechanism of action, the above studies agree that Mtb DosS senses the presence of oxygen. The conclusive elucidation of the mechanism of redox and hypoxia sensing by DosS and DosT in vivo is an area of interest that requires more research. WhiB proteins as iron–sulfur cluster-based sensors Actinobacteria employ a number of mechanisms and pathways for survival during oxidative stress, such as the thioredoxin system and mycothiol pathways. In addition to these important thiol-specific antioxidant systems, another family of proteins has been recently shown to possess properties similar to those of thioredoxins and mycoredoxins. Interestingly, the proteins of this family, called WhiB proteins, harbor an Fe–S cluster and can bind DNA. WhiB proteins were first reported in 1992 by Davis and Chater [129] as products of a screen for genes involved in Streptomyces sporulation. This study demonstrated that the S. coelicolor whiB mutants formed white spores instead of grey spores (thus the name), suggesting a potential role in the regulation of sporulation [129]. Although the sporulation phenomenon has not been reported for Mtb, seven WhiB-like proteins (WhiB1– WhiB7) are present in Mtb and these proteins are in fact conserved throughout the Actinomycetes [6,130]. Sequence alignment of WhiB proteins revealed the presence of a C-terminal HTH motif, four cysteine residues, and a conserved CXXC motif formed by the middle two cysteines, with the exception of WhiB5, which has a CXXXC motif. To begin deciphering the physiological function of WhiB proteins in mycobacteria, Gomez and Bishai tried to create a knockout mutant of whmD (now known as whiB2) [131]. However, unlike nonessential Streptomyces whiB genes, whiB2 is essential and can be disrupted only through complementation in trans. The conditional knockout of whiB2 exhibits filamentous branched growth resulting from aberrations in septum formation and placement, implicating WhiB2 in the regulation of mycobacterial septum formation and cell division [131]. Other whiB genes (such as whiB3 and whiB7) can be disrupted without the need for complementation in trans [132,133] and WhiB proteins have been reported to be involved in a variety of physiological processes, from thioredoxin-like activity [134] to antioxidant response [135] and transcriptional regulatory activity [136]. The following section describes the roles of WhiB proteins in redox sensing, in detoxifying thiol-specific oxidative stress, and as transcriptional regulators of virulence (Fig. 3). WhiBs as iron–sulfur cluster proteins with protein disulfide reductase activity Pioneering studies by Buttner and co-workers first demonstrated the ability of WhiD of S. coelicolor to bind a [4Fe–4S]2 þ cluster through the four invariant cysteines [137]. This [4Fe– 4S]2 þ cluster is oxygen sensitive and can be converted to a [2Fe– 2S]2 þ cluster or completely destroyed [137] upon exposure to O2. These properties are similar to those of the oxygen sensor FNR of E. coli [78]. Interestingly, the four cysteine residues that harbor the iron–sulfur cluster are essential for the function of WhiD. Along similar lines, Steyn and co-workers have reconstituted and characterized the iron–sulfur cluster of WhiB3. They suggested the potential role of WhiB3 in sensing NO and O2 [138]. Their elegant study identified IscS as a cysteine desulfurase and found it to be the main protein involved in the in vitro assembly of WhiB3’s iron–sulfur cluster. Using 35S-labeled cysteines, they developed a traceable, in vitro reconstitution system for the iron–sulfur cluster in WhiB3. This group also established the requirement for all four cysteines in the coordination of the iron– sulfur cluster. Using EPR spectroscopy, they demonstrated that exposure to O2 leads to conversion of [4Fe–4S]2 þ into [3Fe–4S] þ 1, followed by complete destruction of the iron–sulfur cluster, suggesting that WhiB3 is an oxygen sensor similar to FNR of E. coli. Using EPR studies, they further demonstrated that the iron– sulfur cluster of WhiB3 reacts to NO and leads to formation of a dinitrosyl iron complex (DNIC) and thus could potentially be used S.A. Bhat et al. / Free Radical Biology and Medicine 53 (2012) 1625–1641 1633 Fig. 3. Model depicting redox regulation of WhiB proteins. Under hypoxic condition, the WhiB3 protein harbors a [4Feþ 4S]2 þ cluster synthesized by the iron–sulfur cluster-generating machinery consisting of NifS/SufS/IscS. Upon exposure to normoxic conditions the [4Feþ 4S]2 þ cluster of WhiB3 is oxidized to [3Fe þ4S]1 þ , releasing one Fe2 þ , and continual exposure to oxygen leads to further oxidation of the Fe–S cluster to [2Fe þ2S]2 þ . This oxidation-assisted destruction of the Fe–S cluster leads to conformational changes in WhiB proteins that enhance the DNA-binding activity of WhiB3. The activation of WhiB protein could also be achieved through oxidation of CXXC (as in the case of WhiB1) at the cost of reduction of its substrate GlgB or other proteins (not yet identified) to form a disulfide bond. The [4Fe þ4S]2 þ cluster of the WhiB protein could also react with NO to form DNIC. This interaction with NO also activates some WhiB proteins. The active form of WhiB protein has DNA-binding activity and regulates transcription of pks2, pks3, antibacterial resistance genes, and the erm gene. by Mtb to sense NO. Interestingly, the mutant of WhiB3 displayed diminished rugosity in the colony, suggesting an important role for WhiB3 in the regulation of colony morphology. Furthermore, DWhiB3 was attenuated for growth on medium containing carbohydrates as the carbon source, and in contrast the mutant grew significantly better in medium that contained fatty acids as the sole source of carbon. Because the growth on fatty acids is associated with increased reductive stress, these results suggested that WhiB3 could be a sensor of reductive stress, counter to the in vitro experiments suggesting that its iron–sulfur cluster could act as a nano-switch for sensing the presence of O2 and NO [138]. Further scrutiny of the composition of lipids of the whiB3 mutant suggested that WhiB3 regulated the lipid production in Mtb [136]. It was found that WhiB3 regulates the production of the virulence lipids polyacyltrehaloses (PAT), diacyltrehaloses (DAT), sulfolipids (SL-1), trehalose monomycolates, and trehalose dimycolates [136]. Further examination demonstrated that the WhiB3 protein binds to the promoters of pks2 (required for SL-1 biosynthesis) and pks3 (required for PAT/DAT synthesis) and this binding is dictated by the redox state of conserved cysteines. Under oxidizing conditions the binding is enhanced, whereas reducing conditions disrupt the DNA-binding activity, suggesting that WhiB3 is a sensor of oxidative stress rather than reductive stress [136]. Furthermore, this O2/NO-sensing ability of WhiB in mycobacteria is associated with the upregulation of Pks1/Pks3 during macrophage infection and during persistence [66]. The role of WhiB proteins as an Fe–S cluster-based redox sensor was also supported by a recent study that demonstrated that WhiB4 of Mtb contains an oxygen- and NO-sensitive Fe–S cluster. Interestingly, the WhiB4 mutant displays an altered redox balance with accumulating NADH levels in cytoplasm and is resistant to oxidative stress in vitro and in vivo. This mutant was also found to display hypervirulence in the lungs of guinea pigs [139]. The above-cited literature suggests that the WhiB proteins use their Fe–S cluster and act as redox-sensing proteins; however, Agrawal and co-workers [140,134] have attributed a thiol-specific antioxidant property to the WhiB proteins. They cleverly demonstrated that WhiB1 and WhiB4 proteins possess protein disulfide reductase activity and that this activity is regulated through an iron–sulfur cluster. No protein disulfide activity was seen in the case of WhiD of S. coelicolor [141]. This property of reducing disulfide bonds of proteins was attributed to the CXXC motif and was established using the insulin disulfide reductase assay, in which reduction of the disulfide bond between the two chains of insulin leads to precipitation of the b chain and a subsequent increase in OD650. It is likely that these proteins also act as protein disulfide reductases in vivo. If they do, the following question arises: after WhiB1 and WhiB4 reduce the disulfide bonds of their substrates, how are the oxidized WhiB proteins converted back to their reduced form? The Agrawal group showed that the existing thioredoxin reductase cannot reduce WhiB4 [140], but they suggested that the WhiB proteins could function as ‘‘mycoredoxins,’’ using the reductive energy from the NADPH channeled through mycothiol. This hypothesis that WhiBs are mycoredoxins capable of binding iron–sulfur clusters was supported by the discovery that some glutaredoxins can also bind iron–sulfur clusters [142]. Mycoredoxins were recently identified and characterized in Corynebacterium glutamicum by Messens and coworkers [143]. The WhiB proteins, if acting as mycoredoxins, would depend on MSH, and the phenotype of MSH-deficient mycobacteria should be the same as that of the whiB mutant. Contrary to this, no such phenotype has been reported for the MSH-deficient mutant, suggesting that some other uncharacterized proteins could function as mycoredoxins [143]. Recently, Agrawal and co-workers used a yeast two-hybrid screen to identify the a-(1,4)-glucan branching enzyme (GlgB) as the 1634 S.A. Bhat et al. / Free Radical Biology and Medicine 53 (2012) 1625–1641 interacting partner of WhiB1 [144], but more research is needed to find other interacting partners and to establish the mechanism by which WhiB obtains reducing equivalents. Furthermore, a recent study has suggested that WhiB2 has chaperone-like activity, adding a new dimension to the function of WhiB proteins and raising the possibility that WhiB proteins could be multifunctional proteins [145]. WhiBs as redox-responsive transcriptional factors Since their discovery in actinobacteria, WhiB proteins have been postulated to be DNA-binding proteins. WhiB3 was known to physically interact with the principle s factor SigA (RpoV) [133], but the first evidence of its DNA-binding activity was provided only recently by Singh et al. [136], who conclusively demonstrated that WhiB3 can bind specifically to the putative promoter sequences of the lipid biosynthesis genes pks2 and pks3. They also established that oxidized apo-WhiB3 possesses a stronger affinity for DNA than holo-WhiB3 or reduced apo-WhiB3, suggesting an important redox-sensitive switch for DNA binding and transcriptional regulation [136]. Earlier work by the same group established that WhiB3 is involved in O2 sensing. They used EPR spectroscopy to demonstrate that oxygen catalyzes the oxidation of the redox-responsive [4Fe–4S]2 þ cluster to form a [3Fe–4S] þ species and that for WhiB3 to function as a DNAbinding protein, the iron–sulfur cluster is ultimately destroyed [138]. Another recent report by Smith et al. [146] identified a 37bp region of protection by WhiB1 protein in DNase I footprinting assays. This group demonstrated that the iron–sulfur cluster of WhiB1 is oxygen tolerant and reacts rapidly with NO to form dinuclear dinitrosyl-iron thiol complexes. They also showed that holo-WhiB1 (with [4Fe–4S]2 þ ) has lower affinity for its specific DNA than apo-WhiB1 (lacking the iron–sulfur cluster) or holoWhiB1 treated with NO [146]. Furthermore, DNA-binding activity has been detected in WhiB2 and WhiBTM4 (a WhiB homolog) of mycobacteriophage TM4 [147]. Another recent study demonstrated that the WhiB4 of Mtb is capable of nonspecifically binding to GC-rich regions of DNA and this binding leads to repression of transcription. It was further demonstrated that the oxidizing conditions that convert the holo-form of WhiB4 into the apo-form enhance the binding of WhiB4 with DNA and hence induce the repression of transcription [139]. Taken as a whole, the above-cited literature suggests that DNA-binding activity is a common feature among WhiB proteins, but more research is needed to establish that WhiB proteins are iron–sulfur clusterbased nano-sensors of O2 and NO. WhiB proteins as virulence and stress response factors The first evidence of the importance of WhiB3 in Mtb virulence was provided by Steyn et al. in 2002 [133]. Using a yeast twohybrid screen, this group identified WhiB3 as a virulence factor that interacts with RpoV from the virulent strains of Mtb but not with the mutated (Arg515His) allele from the avirulent strain. The interaction of WhiB3 with s factors was suggestive that WhiB3 could function either as a transcription factor or an anti-s factor. It was later demonstrated that WhiB3 harbors an O2- and NOsensitive iron–sulfur cluster that can regulate the expression of stress- and virulence-associated genes in response to redox stress [136,138]. Importantly, a whiB3 mutant of H37Rv shows no growth defect in the murine and guinea pig models of TB, whereas the whiB3 mutant of Mycobacterium bovis is attenuated in both models. These observations suggest that the role of WhiB3 is distinct in different species of pathogenic mycobacteria, in particular Mtb and M. bovis [133]. The expression of WhiB2 and WhiB3 of Mycobacterium avium is upregulated during oxidative stress and pH stress, suggesting a role for these proteins in sensing and responding to these stresses [148]. Moreover, in mouse lungs and macrophages, increased expression of WhiB3 is observed during early infection. The WhiB3 expression profile is dependent on cell density and indicates its regulation by ‘‘quorum sensing’’ [149]. WhiB7 is involved in regulating many antibacterial resistance genes in mycobacteria; it is induced by erythromycin, tetracycline, capreomycin, streptomycin, and kanamycin. In contrast, the whiB7 mutant is hypersensitive to these antimicrobial agents, suggesting that WhiB7 could be a potent chemotherapeutic target [132,150,151]. A recent study by Thompson and co-workers reported that WhiB7 responds to the level of MSH/MSSM, is induced in a reducing environment, and thus is directly involved in the regulation of redox homeostasis of the cell [153]. This study also demonstrated that the macrolide resistance mediated by erythromycin ribosome methyltransferase (ERM) [152] is actually mediated by WhiB7 acting as a transcription factor for the erm gene [153]. Further investigation of the regulatory roles of WhiB proteins in transcription and redox detoxification will provide us a better understanding of their physiological function. Redox-regulated r factors Sigma (s) factors are the primary regulators of bacterial gene expression. The Mtb genome encodes 13 members of the s70 family [6]. SigA, SigB, and SigC belong to groups 1, 2, and 3, respectively, and the rest belong to group 4. Group 4 s factors are involved in sensing extracytoplasmic signals; these proteins are also called extracytoplasmic function s factors [154]. s factors regulate transcriptional responses for numerous cellular processes in prokaryotes, including stress response and growth. Normally, s factors can recognize specific promoter sequences and interact with components of RNA polymerase, but they cannot directly sense relevant specific signals. Instead, they are guided by anti-s factors, anti-anti-s factors, and a variety of sensors and transcription regulators to synchronize the appropriate transcriptional response to stress. SigH, SigE, SigL, and SigF play important roles in the survival of Mtb against redox stress; their mechanism of redox sensing is described below (Fig. 4). The role of SigH in oxidative stress was first demonstrated by Fernandes et al. using M. smegmatis sigH mutants [155]. These mutants are extremely susceptible to cumene hydroperoxide, suggesting that SigH is important for the regulation of mycobacterial response to oxidative stress. The same group later demonstrated that SigH is a homolog of SigR of Streptomyces and plays an important role in the protection of Mtb from oxidative thiol stress and ROS [156,157]. It was shown that SigH protects against oxidative stress by regulating the expression of thioredoxins (trxB1 and trxC), thioredoxin reductase, and stress-responsive s factor SigE (Fig. 4A). SigE and SigH regulate the expression of stress-responsive s factor SigB. Further evidence for the protective role of SigH comes from the observation that it is upregulated upon infection of macrophages [158]. Interestingly, infection of mice with a sigH mutant of Mtb [159] produces an organ burden similar to that of mice infected with wild-type Mtb. However, the immunopathology of animals infected with sigH mutant strains is nominal compared with that of mice infected with wild-type Mtb, suggesting that SigH regulates the immunopathogenic virulence factors of Mtb [159]. Additionally, ingenious studies performed by Song et al. have demonstrated that Rv3221a, a gene in the same operon as sigH, codes for a bona fide anti-s factor against SigH [160], named RshA. RshA interacts specifically with SigH in a 1:1 stoichiometry in vitro and in vivo [160] and the interaction of RshA with SigH leads to inhibition of SigH-dependent transcription S.A. Bhat et al. / Free Radical Biology and Medicine 53 (2012) 1625–1641 1635 Fig. 4. Response of s factors and anti-s factors to redox stress. The activity of s factors is regulated by an intricate network of anti-s factors and anti-anti-s factors. The binding of an anti-s factor inhibits the activity of s and is regulated as shown in (A) and (B). (A) Redox regulation of DNA-binding activity of SigH and SigE. SigH regulates the expression thioredoxins (trxB, trxC) and thioredoxin reductase (trxR), which constitutes a vital response to oxidative stress. Under stress-free conditions, anti-s factor RshA binds SigH and inhibits the transcription of thioredoxins, thioredoxin reductase, and sigE. The release of SigH from anti-s factor RshA is achieved under oxidative stress by two mechanisms: (i) under oxidative stress the Zn2 þ bound to the cysteines and histidine of RshA is released to form a disulfide bond, resulting in conformational change in RshA leading to release of SigH. (ii) Phosphorylation of RshA by PknB renders it inactive and activates SigH. In addition to activation by SigH, the sigE transcriptional activation is also regulated by polyphosphate (poly-P) stress. Poly-P activates the MprAB two-component system, which regulates transcription of SigE. However, the activity of SigE is also regulated posttranslationally through anti-s factor RseE. SigE is released from RseA by two mechanisms: (i) under oxidative stress the redox-reactive cysteines of RseA undergo oxidation to form a disulfide bond and induce conformational changes guiding the release of SigE. (ii) SigE leads to transcriptional activation of relA, clgR, and sigB. ClgR upregulates the ClpC1P2 system. This ClpC1P2 binds to phosphorylated RseA (phosphorylated by PknB) and RseA is cleaved by these proteases, activating SigE. SigE also regulates its own transcription. (B) Redox-mediated regulation of SigF. Antibiotics, stationary phase, anaerobiosis, and oxidative stress lead to activation of sigF and anti-s factor usfX transcription. SigF is involved in persistence and possesses a complex control mechanism involving anti-anti-s factors RsfA and RsfB. One mechanism involves the binding of RsfA to UsfX under reductive stress in the stoichiometry 1:2. This binding of RsfA to UsfX releases and activates SigF. The other mechanism involves the anti-anti-s factor RsfB, which becomes phosphorylated by an unknown kinase and releases SigF to activate the genes involved in persistence of the bacteria under oxidative stress. in vitro. The peptides involved in RshA–SigH interactions were identified through a phage display screen. These peptides inhibited SigH by mimicking RshA [161]. More importantly, the interaction was sensitive to oxidative stress and heat shock [160]. Similar to RsrA of Streptomyces, RshA harbors an HXXXCXXC motif. This motif, called the ZAS motif, is found in a number of redox-sensitive, zinc-associated anti-s factors. The cysteine thiols of the ZAS motif bind Zn2 þ under reducing 1636 S.A. Bhat et al. / Free Radical Biology and Medicine 53 (2012) 1625–1641 conditions; under oxidizing conditions, these redox-active cysteines form a disulfide bond and release the zinc [162,163]. When the cysteines of the HXXXCXXC motif in RshA were mutated to alanine, the inhibitory effect of RshA on transcription by SigH was abolished. Furthermore, oxidation of these conserved cysteine residues inhibited the binding of RshA with SigH [160], thus ensuring induction of a protective response during oxidative stress. Another important s factor that regulates mycobacterial response to oxidative stress is SigE, as evident from the observation that sigE knockout strains of M. smegmatis are more susceptible than wild-type strains to oxidative stress and to several membranedisrupting agents [164]. Further experiments showed that sigE expression is induced upon phagocytosis of macrophages [165] and that Mtb mutants of sigE are defective in survival against oxidative stress and inside the macrophages [166]. Additionally, SigE is important in Mtb virulence in the mouse model of TB [167,168]. The two-component system MprAB, which responds to polyphosphate stress [169] and is implicated in the persistence of Mtb in vivo [170], was shown to regulate the expression of SigE. Interestingly, SigE regulates its own expression and directly regulates the level of RelA and MprAB, thereby creating a feedback loop. RelA is important for long-term survival of mycobacteria under starvation conditions [171]. SigE also regulates the expression of clgR, sigB, and a number of other genes involved in the survival of Mtb under stress conditions [166]. Importantly, the function of SigE is regulated by the anti-s factor RseA, which has a ZAS motif (Fig. 4A). RseA interacts with and inhibits the function of SigE under reducing conditions. This interaction requires Cys70 and Cys73 of the ZAS motif and is disrupted by oxidizing conditions, suggesting that the disulfide bond formation between the two cysteines acts as the regulatory step of the interaction [172]. Another level of complexity in the regulation of SigE function is introduced by phosphorylation of RseA on Thr39 by PknB in response to surface stress-imparting agents such as vancomycin. This phosphorylation renders RseA susceptible to proteolysis by the SigE-regulated ClpC1P2 proteolytic machinery. This signaling constitutes an envelope stress-induced positive feedback loop for SigE-dependent transcription [172]. Another redox-regulated s factor is SigL of Mtb. SigL is cotranscribed with and binds specifically to RslA (Rv0736). RslA is predicted to be a membrane protein, wherein the ZAS motif on the N terminus of the protein faces the exterior of the cell and the C-terminal anti-s factor domain resides in the cytosol. SigL regulates a number of cell envelope proteins, suggesting its importance in TB pathogenesis. This idea is supported by the observation that mice infected with a sigL mutant of Mtb survive longer than mice infected with wild-type Mtb [173]. Elegant studies performed by Thakur et al. have elucidated the mechanism of regulation of SigL through the redox sensor RslA [174]. This group solved the crystal structure of the SigL–RslA complex and observed that under reducing conditions, the C-terminal anti-s factor domain of RslA binds SigL and the N-terminal domain coordinates Zn2 þ through His25, His50, Cys54, and Cys57. RslA inhibits transcription by SigL through the occlusion of the s4 domain, which recognizes the 35 region of SigL-specific promoters. Oxidative stress assists the release of Zn2 þ through formation of a disulfide bond between the cysteine residues of the ZAS motif. This drives a conformational change in the anti-s factor domain of RslA and reduces its affinity for SigL, thus permitting transcription activation by SigL [174]. Recently obtained evidence links SigF with redox stress. SigF is a homolog of bacillus s factor, which regulates sporulation in response to nutrient and amino acid starvation. SigF is induced upon antibiotic exposure, in the stationary phase of growth, upon anaerobiosis, and in response to oxidative stress [175], suggesting a role for SigF in Mtb redox homeostasis. Importantly, the SigF mutant is attenuated for persistence in the murine model of TB [176]. SigF regulates a number of genes involved in stability of the cell envelope, including sulfolipids [176]. SigF itself is regulated by the anti-s factor UsfX (Rv3287c). usfX is located upstream of sigF in the Mtb genome and is transcribed as an operon under the control of SigF [177] (Fig. 4B). Well-executed studies by Beaucher et al. demonstrated that UsfX specifically binds and inhibits transcription by SigF [178]. This study further identified two anti-anti-s factors: RsfA (Rv1365c) and RsfB (Rv3687c). RsfA binds specifically to UsfX and relieves SigF inhibition under reducing conditions. This binding depends on Cys73 and Cys109. RsfB, however, seems to be regulated through phosphorylation of Ser61. Interestingly, Rv1364c also shares significant sequence similarity with the anti-anti-s factors of SigF, but formal evidence that Rv1364c regulates SigF is lacking. Importantly, Rv1364c has a PAS domain with very high affinity for fatty acids (particularly palmitoleic acid), suggesting that it may regulate SigF in response to fatty acids [179]. In summary, a complex network of s factors and their regulatory protein partners ensures survival of Mtb during physiological redox stress. The role of serine–threonine kinases in redox homeostasis of mycobacterium In most eukaryotes, signal is perceived by a sensor and then relayed through an intricate network of kinases and phosphatases to generate a synchronized response. The Mycobacterium genome encodes 11 serine/threonine kinases (STKs) (PknA to PknL, except PknC), one serine/threonine phosphatase (PstP), a tyrosine kinase (PtkA), and two tyrosine phosphatases (PtpA and PtpB). These kinases and phosphatases, along with two-component systems and onecomponent proteins, constitute the signal sensing and transducing machinery of Mtb [180]. Knowledge of the molecular basis of this signal transduction network lies at the heart of our ability to create novel therapeutic tools; our current understanding of the roles of kinases in the redox homeostasis of Mtb is described here (Fig. 5). PknA and PknB are encoded by an operon located near the origin of replication. This operon also codes for PstP, RodA (a protein involved in determination of cell shape), and PbpA (a protein involved in peptidoglycan biosynthesis). PknA and PknB are essential genes that are involved in the regulation of cell growth and morphology. PknA can phosphorylate PknB, Wag31, FipA, FtsZ, and FtsQ [174,181]. Wag31, FipA, FtsZ, and FtsQ are involved in cell division. Interestingly, phosphorylation of FipA on Thr77 and FtsZ on Thr343 by PknA is required for cell growth under oxidative stress [181], suggesting an important role for PknA in sensing oxidative stress and regulating oxidative stress-responsive growth. PknA is sufficiently different from human kinases and thus is an attractive drug target. With the aim of discovering novel anti-TB drugs, Magnet et al. [182] have devised a screen for identifying PknA inhibitors [164]. Of the 12,000 compounds that were screened, three potential hits with a minimum inhibitory concentration below 10 mM were identified [182]. PknB phosphorylates the oxidative response s factor SigH and its anti-s factor RshA. Phosphorylation of RshA by PknB leads to disruption of the interaction between SigH and RshA and thus regulates the induction of oxidative stress response by mycobacteria [183]. Similar to PknB, PknD indirectly regulates the transcriptional response of SigF through phosphorylation of the anti-anti-s factor Rv0516c. However, this phosphorylation differs from classical phosphorylation on a conserved internal threonine and is located on the second amino acid at the N terminus (Thr2) [184]. Two of the STKs (PknG and PknE) harbor redox-sensitive domains, suggesting that these STKs could be important for sensing the redox environment of mycobacteria. Of these, PknG S.A. Bhat et al. / Free Radical Biology and Medicine 53 (2012) 1625–1641 1637 Fig. 5. Protein kinases as redox sensors. Top left: PknA phosphorylates PknB, Wag31, FtsQ, FipA, and FtsZ. They are involved in the control of cell growth and morphology; among these proteins FipA and FtsZ are very important as they are involved in cell growth under oxidative stress. Phosphorylated PknB also activates SigH by phosphorylating the anti-s factor RshA. Top right: PknG activates a-ketoglutarate decarboxylase and glutamate dehydrogenase by phosphorylation of inhibitory protein GarA. Bottom: PknD is involved in activation of SigF by phosphorylating and inhibiting the binding of anti-s factor Rv0516 to SigF. is of special interest and is studied vigorously. PknG inhibits the phagosome–lysosome fusion of infected macrophages and thus plays an important role in mycobacterial virulence [185]. The inhibition of PknG’s kinase activity by chemical inhibitors leads to increased clearance of mycobacterial infection, suggesting that PknG could be exploited as a potential drug target [185]. PknG has an N-terminal rubredoxin domain, a central kinase domain, and a C-terminal tetratricopeptide repeat domain. Rubredoxin domains are small domains that bind iron tetrahedrally through four cysteine residues that reside in two CXXCG motifs. They are usually found in electron carrier proteins, but the rubredoxin domain in PknG, a sensor STK, is probably a redox sensor. The rubredoxin domain is important for regulating the function of PknG [186,187]. The kinase activity of PknG is induced in response to oxidative stress and the cysteines of the CXXCG motif are closely involved in regulating that kinase activity [187]. Additionally, PknG leads to phosphorylation of GarA (glycogen accumulation regulator) in Mtb [188]. Unphosphorylated GarA binds and inhibits a-ketoglutarate decarboxylase and glutamate dehydrogenase and thus regulates metabolic flux in response to nitrogen availability [188]. PknE harbors a thioredoxin fold. This fold is often present in redox sensors, indicating that PknE might act as a redox sensor. Interestingly, expression of PknE is responsive to NO and deletion of PknE leads to increased resistance to NO and increased sensitivity to reducing agents [189]. Similar to PknE, deletion of PknH in Mtb also leads to increased resistance to acidified NO, implying that PknH could also act as a redox sensor. Recently, AvGay and co-workers demonstrated that PknH phosphorylates the NO-responsive DosR [190] on Thr198 and Thr205. These two phosphorylations and the phosphorylation by DosS and DosT on Asp act cooperatively to enhance the transcription activation by DosR in response to NO [190]. In summary, the STKs of Mtb are important for sensing redox stress and orchestrating an ordered response to the stress. in vitro leads to induction of specific transcriptional responses that result in networked regulation of Mtb’s metabolism facilitating the maintenance of intracellular redox state and survival. The induction of specific transcriptional responses despite a lack of classical redox sensors suggests that Mtb possesses a battery of redox sensors to steadily monitor the extracellular and intracellular redox status. Some of these sensors have been recently discovered; however, many more redox sensors are yet to be discovered. The important redox sensors of Mtb include the heme based sensors DosS and DosT, Fe–S cluster-based WhiB proteins, redox-responsive thiolbased anti-s factors/anti-anti-s factors, and thioredoxin-, rubredoxin-fold-dependent redox-sensitive kinases. The DosS and DosT proteins monitor changes in the levels of oxygen, redox, NO, and CO to facilitate the transition from an actively multiplying state to a nonreplicative state. The WhiB proteins sense changes in the NO, oxygen, and intracellular redox state to regulate the metabolism of virulence lipids and response to antibiotics. The anti-s/anti-antis factors and serine–threonine kinases monitor the levels of ROS. However, we still do not know the identity of the sensors involved or the genetic pathways involved in reactivation of Mtb upon reaeration/dampened immune system. Thus many more sensors and regulatory pathways await discovery. Given the unique and prolonged latency of Mtb, these pathways and sensory modules would be unique to mycobacteria and could be exploited for discovering drugs that may target the bacteria in a persistent/latent infection. Acknowledgments We thank Dr. Girish Sahni for his help and support. We gratefully acknowledge IMTECH (a constituent laboratory of the CSIR) for providing the infrastructural facilities and financial support. A.K. is supported by funding from the CSIR (OLP-70 and SIP10) and DBT (BT/PR15097 and BT/PR15086/GBD/27/307/ 2011) India. S.A.B., N.S., and P.K. acknowledge the fellowships from CSIR and A.T. is grateful to UGC for a fellowship. Concluding remarks References Mtb is persistently exposed to numerous redox stresses during its pathogenic cycle. These stresses include host-generated ROS, acidic pH, RNS, and hypoxia. Exposure of Mtb to these stresses [1] World Health Organization. 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